by phillips w. rcbbinst and generallowing: 1000 mmoles of imidasole*hcl, ph 7; 100 pmoles of...

ISOLATION AND IDENTIFICATION OF ACTIVE SULFATE*

BY PHILLIPS W. RCBBINSt AND FRITZ LIPMANNf

(From the Biochemical Research I.aboratory, Massachusetts General Hospital, and the Department of Biological Chemistry, Harvard Medical School,

Boston, Massachusetts)

(Received for publication, July 26, 1957)

In an earlier paper (l), indicative although preliminary results were obtained in the attempt to characterize the mechanism of ATP-linked’ sulfate activation, It had been found that pyrophosphate is a product of the reaction. The earlier observations (2, 3) on the two-phasic nature of the over-all process of phenol conjugation were confirmed and a preliminary separation was obtained of the enzyme fractions responsible for the activation of sulfate and the transfer of active sulfate to phenol. Although sizable amounts of the active sulfate could not be obtained at that stage, it was concluded from paper electrophoresis patterns that the compound must be a sulfate derivative of adenylic acid, the exact chemical composition of which, however, remained to be decided.

One of the handicaps in these experiments had been the lack of a reli- able assay method for active sulfate applicable to cruder mixtures, in particular, those containing ATP in addition to the active sulfate. In view of the difficulties for chemical determination of sulfate in micromolar quantities, the enzymatic assay with nitrophenol as acceptor proved by far the easiest way to determine the activated compound. In order to obtain an assay system for crude preparations, however, it became essential to separate the sulfate-activating system from the transferring enzyme which we designate here as phenol sulfokinase. After this assay system was developed, the experiments leading to the final identification progressed more rapidly. Important, furthermore, was the applicability of the well developed chromatography on Dowex 1 (4). The compound proved to be not as fragile as originally thought but relatively stable, even

* This investigation was supported in part by a research grant from the National Cancer Institute, National Institutes of Health, United States Public Health Serv- ice, and the Life Insurance Medical Research Fund.

t Fellow of the National Foundation for Infantile Paralysis. Present address, The Rockefeller Institute, New York 21, New York.

$ Present address, The Rockefeller Institute, New York 21, New York. 1 The following abbreviations are used: PAPS for 3’-phosphoadenosine-5’-phos-

phosulfate; PAP for 3’,5’-diphosphoadenosine; p-NP and p-NPS for p-nitrophenol and its sulfate ester; AMP, ADP, and ATP for adenosine mono-, di-, and triphos- phates; Tris for tris(hydroxymethyl)aminomethane; CoA for coenzyme A; TCA for trichloroacetic acid; and l for molecular extinction coefficient.

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838 ACTIVE SULFATE

to the rather high concentrations of formic acid which had to be used in its chromatography. It has already been reported in a preliminary communi- cation (5) that the active sulfate now has been identified as adenosine 3’- phosphate-5’-phosphosulfate.’

Materials and Methods

p-NP was a product of the Eastman Kodak Company and was purified by recrystallization from water. Enzymatic formation of p-NPS was followed by measuring the disappearance of nitrophenol by the method of Hilz and Lipmann (1). This method depends upon the calorimetric determination of nitrophenol at 400 rnp in alkaline solution. Nitrophenyl sulfate gives no color under these conditions. The active sulfate, PAPS, was determined by transfer to nitrophenol as described below or by the PAP assay (6). Inorganic phosphate was determined by the method of Fiske and Subbarow (7), and total phosphate after ashing with HzSOa (8). Sulfate was determined according to the method described by Fromageot (9).

Phosphate labeled with P32 and Sa6-labeled sulfate were obtained from the Oak Ridge National Laboratory. P3*-labeled ATP was prepared by the tryptophan-catalyzed P32-pyrophosphate exchange with tryptophan- activating pancreas enzyme (10). Bull semen 5’-nucleotidase and 3’- nucleotidase were generously supplied by Dr. N. 0. Kaplan. The nucleotidase was subsequently prepared from rye grass seed (11) according to Shuster and Kaplan (12).

Titrations were madewitha model TTT/l Radiometer automatic titrator. Saturated (NHJ2SOd was prepared as described previously (1) and crystalline ATP was purchased from the Pabst Laboratories. An apparatus similar to that described by Durrum (13) was used for paper electrophoresis. Protein concentrations were determined turbidimetrically with trichloroacetic acid (14) and refer to crystalline bovine serum albumin as a standard.

Enzyme Preparations

Separation of Sulfate-Activating System and Phenol Suljokinase-Rabbit liver or fresh lamb liver was cut into pieces and chilled in ice. Lamb livers which have been allowed to stand at room temperature for 30 minutes or more are usually inactive. The preparation was carried out, as described previously (l), through the removal of the microsomes. This extract may be frozen and stored without loss of activity.

To 250 ml. of liver supernatant fluid were added 100 ml. of C-r alumina gel (cf. Hilz and Lipmann (l)), containing 15 mg. of dry material per ml., and the mixture was stirred for 30 minutes at 04”. All subsequent steps


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P. W. ROBBINS AND F. LIPMANN 839

were carried out at 04”. The gel was separated from the solution in the International centrifuge at 5000 r.p.m. for 5 minutes. The gel supernatant fluid contains the phenol sulfokinase free from the sulfate-activating system. It may be stored in the deep freeze without loss of activity. In earlier experiments (1) adsorption of the sulfate-activating system on the gel appears to have been less efficient and much of it seems to have leaked into the supernatant fluid. With the present procedure, however, it has been rather consistently found that the sulfate-activating system is completely adsorbed to the alumina gel. In order to avoid loss it should be eluted immediately.

Sulfate-Activating System-The gel was washed by resuspension in 250 ml. of 0.1 M (NH&S04 and was stirred for 30 minutes. It was then centrifuged and the supernatant fluid discarded. The gel was resuspended in 200 ml. of 0.8 M (NH&SO4 and extracted by stirring for 60 minutes. After centrifugation, saturated (NH&SO4 was added to the alumina gel extract to a final saturation of 60 per cent. The precipitated protein was collected in the SS-1 Servall centrifuge and dissolved in 0.02 M Tris, pH 7.5, to give a protein concentration of 10 to 20 mg. per ml. This preparation will synthesize PAPS from ATP at a rate of about 0.2 pmole per mg. of protein per hour. A further extraction of the gel with 0.8 M (NH&SO4 gives additional enzyme of a somewhat higher specific activity.

Phenol Sulfolcinase-Saturated (NHd)&Od was added to the original alumina gel supernatant flmd to a final saturation of 55 per cent. After standing for 2 hours, the solution was centrifuged for 15 minutes in the Servall SS-1 centrifuge and the precipitate was dissolved in 0.02 M Tris, pH 7.5. This preparation contains the transferring enzyme almost completely free from sulfate-activating enzyme. Phenol sulfokinase may be assayed most conveniently by the method of Gregory and Lipmann (6). This assay depends on the transfer of sulfate from p-NPS to phenol in the presence of PAP and phenol sulfokinase. The transferring enzymes have recently been fractionated more extensively (6, 15).

Assay of PAPS-Active sulfate was assayed either by direct transfer to nitrophenol or by the PAP-PAPS assay described in another paper from this laboratory (6). The assay system for the direct transfer reaction contained 100 pmoles of imidazole hydrochloride, pH 7.0, 10 pmoles of cysteine, 1 pmole of p-nitrophenol, and 3 mg. of the transferring enzyme in a final volume of 1 ml. Nitrophenolate disappearance was determined after 30 minutes incubation at 37”. The initial concentration of PAPS was usually 0.5 pmole per ml. or less. Otherwise, the transfer was in- complete because the reverse reaction, nitrophenyl sulfate plus PAP, be- comes appreciable (6). Gregory and Lipmann have shown that the


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840 ACTIVE SULFATE

apparent equilibrium constant for the transfer of sulfate from PAPS to p-NP is approximately 26.

Kapp - bNPS1 X [PAP1 = 26 [p-NPI x [PAPS]

Therefore, if 50 per cent of the p-NP has reacted with PAPS to form p- NPS at equilibrium, the analytical error in the determination of PAPS will be 4 per cent. When more than 50 per cent of the p-NP is converted to p-NPS, the analytical error will become appreciable. Actually, additional losses are introduced by the presence of interfering enzymes in the phenol sulfokinase preparation such as phosphatases which attack PAPS and sulfatases which hydrolyze p-NPS. Even under the most favorable conditions the transfer of sulfate from PAPS to p-NP was seldom more than 90 per cent complete. This relatively small deficit could generally be neglected. The great simplicity of the assay outweighs an error small enough to be generally of no importance. In assaying enzymatic reaction mixtures for PAPS, protein was removed by boiling for 2 minutes and centrifugation, and the supernatant fluid was added directly to the transferring reaction mixture. No inhibition was found in the transfer reaction due to the ATP, inorganic sulfate, and MgClz present in the incubation mixture. The requirement of the transferring enzyme for cysteine or glutathione is discussed elsewhere (6). Fig. 1 shows the time-course for the transfer of sulfate from PAPS to nitrophenol. Transfer is practically complete after 30 minutes.

The use of the PAP assay for active sulfate determination depends on the catalytic activity of PAP for transfer of sulfate from nitrophenyl sulfate to phenol (6). In this assay the catalytic function of PAP consists in operat- ing as obligatory sulfate carrier. The assay therefore does not discriminate between PAP and PAPS but is equally applicable to both.

Assay of Sulfate-Activating System-The PAPS assays described above may be used conveniently for determining the activity of the sulfate-activating enzyme system. Incubations are carried out as described below and the reaction is stopped by boiling. The amount of PAPS formed in 30 minutes is proportional to the enzyme concentration.

I soldion of PAPS

The incubation mixture used for the accumulation of PAPS contained 0.1 M imidazole hydrochloride, pH 7.0, 0.0125 M MgCh, 0.02 M K2S04, 0.01 M ATP, 0.01 M cysteine, and 2 to 5 mg. per ml. of protein from the alumina gel extract. Fig. 2 shows the rate of PAPS accumulation under these conditions.

In preliminary experiments for the isolation of PAPS from Dowex 1 columns, the method of Siekevitz and Potter (4), a modification of the


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procedure of Cohn and Carter (16), was used. A deproteinized incubation mixture (10 ml.) containing 0.3 pmole per ml. of transferable sulfate was adjusted to pH 8.0 with 2 N KOH and applied to a 1 X 3 cm. Dowex 1 formate column. The column was operated at a temperature of 2”. Fractions were eluted with 60 ml. volumes of 0.01 M NH, formate, 2 N

formic acid (Fraction I), 3 N formic acid (Fraction II), 4 N formic acid-O.3 N NH, formate (Fraction III), and 5 N formic acid-l N NH, formate (Frac- tion IV). The fractions were lyophilized, dissolved in 5 ml. of water, and

IO 20 30 40 50 60 IO 20 30 40 50 T/ME /Iv MINUTES T/M- /N MhVUTES Fxa. 1 FIN. 2

Pro. 1. Transfer of PAPS to p-NP. The incubation mixture contained the following: 1000 Mmoles of imidasole*HCl, pH 7; 100 pmoles of cysteine; 10 pmoles of nitrophenol; and 30 mg. of phenol sulfokinase in addition to PAPS in a final volume of 10 ml. Incubation at 37’.

FIQ. 2. Accumulation of PAPS by liver sulfate-activating system. The reaction mixture is described in the text. Incubation wae at 37”. Samples (1 ml.) were taken at the time intervals indicated, heated in boiling water for 2 minutes, then chilled and centrifuged. To exactly 0.3 ml. of the supernatant solution was added 0.2 ml. of a mixture made by adding 0.5 ml. of 0.01 Y p-NP to 1.5 ml. of phenol sulfokinase. Nitrophenol disappearance was measured after 30 minutes incubation at 37”.

neutralized with 2 N KOH. Since the phosphosulfate link is alkali-stable, no particular care needs to be taken with this neutralization. Fig. 3 shows the paper electrophoresis pattern and transferable sulfate content of the column fractions. Fraction IV represented at least 70 per cent of the active material placed on the column. The adenosine concentration as determined by absorption at 260 ~QI was 0.75 pmole per ml.; a trace of ATP was still present in the active fraction.

In subsequent larger scale preparations of the PAPS fraction, 40 ml. of protein-free incubation mixture were applied to a 1.6 X 10 cm. Dowex 1 formate column after dilution to 80 ml. with cold water and adjustment of the pH to 8. The sample was followed with 50 ml. of cold distilled


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842 ACTIVE SULFATE

water. Elution with 4 N formic acid-O.3 N NH4 formate was started immediately. Usually about 500 ml. of this buffer solution were required to remove the Cl-, POP, SO,-, AMP, ADP, and ATP from the column. Elution was continued until the optical density at 260 rnp fell well below 0.5. Then PAPS was eluted with 5 N formic acid-l N NH4 formate. Large fractions (20 ml.) were collected, and the three or four having the highest optical density at 260 rnp were pooled and lyophilized. Since PAPS is acid-labile, the collection and lyophilization were carried out aa

PAPS-, 0 ;g -c + 0 0 0 ..__-’ .,---, z

ADP--0 0 0

OR/G/At 1 I I I I I I I STANDARDS I FRACTION I

rlrlum TRANSFERABLE

SULFATE - f

0.00 0.00 0.00 0.46 yMOLES/ML

FIG. 3. Paper electrophoresis pattern of Dowex 1 column fractions. The fractions were dissolved in 5 ml. of water and neutralized with 2 N KOH before analysis. One aliquot (0.3 ml.) was taken and tested for enzymatic transfer of sulfate to p-NP. Another aliquot (0.03 ml.) was used for electrophoresis on Whatman No. 31 paper. Electrophoresis was carried out in 0.03 M citrate, pH 5.9, at 2” for 16 hours, with use of 200 volts (3.5 volts per cm.).

rapidly as possible. Even under the best conditions, 20 to 30 per cent of the PAPS was hydrolyzed, largely during lyophilization. In lyophilizing the active fraction, 35 ml. or less were quickly shell-frozen in a 2 liter flask. Lyophilization was limited to 15 to 20 hours. The residue, a mixture of PAPS, PAP, and NH4 formate, was dissolved in a minimum of distilled watek, neutralized with 1 N KOH, and, if necessary, again lyophilized.

Characterization of PAPS

Chemical Stability of Active Sulfate-PAPS is hydrolyzed readily in acid. As shown in Fig. 4, PAPS has a half life in 0.1 N HCl at 37” of about


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6 minutes. The phosphosulfate link is resistant to alkali at room temperature. Incubation at 37” in 0.1 N KOH for an hour gave no detectable loss

60- 60- ‘*. ‘*. 1 1 1 1 I I

0 0 4 4 8 8 12 12 16 16 MINUTES

Fra. 4. Hydrolysis of PAPS in 0.1 N HCl at 37“. A solution of PAPS isolated by chromatography on Dowex 1 was warmed to 37” and then made 0.1 N with respect to HCI. Aliquots (0.25 ml.) were taken and added to a mixture of 255 pmoles of KOH; 50 Imoles of imidasole.HCl, pH 7; and 0.5 rmole of p-NP in 0.1 ml. After mixing, 0.05 ml. of 0.1 M cysteine.HCI and 0.1 ml. of phenol sulfokinase were added to give a final volume of 0.5 ml. The disappearance of p-NP was measured az in Fig. 2.

TABLE I Analytical Data for Active Sulfate Fraction

Adenosine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Total phosphate. . . . . . . . . . . ....... . . . . . ...... 1.98 12 min. phosphate.. . . . . . . . . ....... . . . . . ...... 0.53 30 “ “ . . . . . . . . . ....... . . . . . ...... 1.04 3’-Nucleotidaae phosphate. ....... . . . . . ...... 0.89 PAPS, enzymatic.. . . . . . . . . ....... . . . . . ...... 0.2-0.85 PAPS + PAP.. . . . . . . . . . . . . . . . . . . . . . . . . . . 0.95

I Adenosine was determined by absorption with 6~60 = 14.5 X 10’. It is used aa

reference, assuming it to be 1. The values for 12 and 30 minute phosphate represent the amount of inorganic phosphate released in 1 N HCl at 100”. Phosphate hydrolyzable by 3’-nucleotidase was determined by the method of Wang et al. (18). Since a wide range of values for PAPS hae been observed by the method of isolation described in the text, the range, rather than a particular value, is given for PAPS. The other figures are values obtained by analysis of a single preparation.

of transferable sulfate and heating at 100” for 2 minutes gave no hydrolysis of PAPS in solutions buffered between pH 7 and 9.

AnuZyticuZ Data-Table I shows analytical data for the isolated PAPS fraction. Enzymatic assay showed that a variable fraction of the adenine compounds could be accounted for as transferable sulfate. However, over 90 per cent of the adenine was active in the PAP assay, showing that


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844 ACTIVE SULFATE

this fraction is a mixture of PAPS and PAP, i.e. hydrolyzed active sulfate. Apparently the 5 N formic acid-l M formate eluate represented originally a rather homogeneous solution of PAPS which, during lyophilization, forms more or less PAP by loss of sulfate, depending on the length of ex- posure. As would be expected for a mixture of PAPS and PAP, the phosphate analysis showed consistently two phosphates per adenosine. One phosphate is hydrolyzed in 1 N HCl at 100” after 30 minutes, as is the case with adenosine 2’- or 3’-phosphate (17). The second phosphate is rather acid-stable, corresponding to an adenosine 5’-phosphate.

Hydrolysis of PAPS by S’-Nucleotidase-To identify the position of the more easily hydrolyzed phosphate, 3’-nucleotidase was used. The prepa-

l -ACTIVE SULFATE

Fro 5. Hydrolysis of the active sulfate fraction by 3’nucleotidaee. The incubation tubes contained 100 rmoles of Tris, pH 7.5; 1 ml. of 3’nucleotidaee solution; and 12 amoles of nucleotide in a final volume of 3 ml. Incubation was at 37”. Ali- quots (0.2 ml.) were used for inorganic phosphate determination after stopping the reaction with 1 ml. of cold 10 per cent TCA. The active sulfate fraction was a mixture of PAPS and PAP obtained by chromatography on Dowex 1.

ration and specificity of rye grass 3’nucleotidase have been described by Shuster and Kaplan (11, 12). It has been used to determine the position of the third phosphate group in coenzyme A (18). PAPS is attacked by 3’-nucleotidase at approximately the same rate as CoA. The time-course for the hydrolysis is shown in Fig. 5. At completion more than 90 per cent of the easily hydrolyzable phosphate is released as inorganic phosphate. The products of hydrolysis should be adenosine 5’-phosphosulfate from dephosphorylation of PAPS and AMP from PAP. For verifica- tion, EWPAPS was prepared from labeled inorganic sulfate with use of the method described above for incubation and chromatography. Paz- labeled PAPS was prepared with ATP labeled in the two terminal phosphate groups. Fig. 6 shows a paper electrophoresis pattern for EWlabeled PAPS before and after treatment with 3’-nucleotidase. The radioactive adenosine 5’-phosphosulfate formed was found to have the same electro-


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phoretic mobility as the synthetic compound prepared by the method of Baddiley et al. (19). The electrophoresis pattern shows, furthermore, that PAP is hydrolyzed by the enzyme to AMP. Both synthetic and

ATP

Oo ADP

0

AMP

0 O0

OR/G/N ; i I I

STANDARDS BEFORE AFTER 3 HRS. 3units Gunits

FIG. 6

ATP

0 ADP 0

AMP 0

ORIGIN - STANDARDS BEFORE AFTER 3 HRS.

INCUBATION

FIG. 7 FIG. 6. Hydrolysis of S36-labeled PAPS by 3’-nucleotidase. For preparation of

the PAPS, the incubation mixture contained 2.5ml. of 1 M imidazole, pH 7; 2.5ml.of 0.125 M MgCln; 0.5 ml. of carrier-free radioactive Sod- solution (0.35 mc.); 156 mg. of ATP; 40 mg. of cysteine.HCl; 0.37 ml. of 2 N KOH; and 15 ml. of dialyzed sulfate- activating enzyme system in a final volume of 25 ml. The enzyme preparation was dialyzed for 6 hours against a flowing solution of 2 M KCl-0.05 M KHCOa-0.001 M reduced glutathione. This procedure reduced the sulfate concentration of the enzyme solution from 1.3 M to 0.05 M. Incubation was for 1 hour at 37”. After heating for 2 minutes at 100° and centrifugation to remove denatured protein, chromatography on Dowex 1 was carried out as described in the text. Separate samples of the radioactive PAPS were incubated with 3 and 6 units of 3’-nucleotidase for 3 hours according to the procedure of Wang et al. (18). Aliquots of the original solution and of the hydrolysis reaction mixtures were used for paper electrophoresis in 0.03 M citrate, pH 5.9. Outlined areas represent ultraviolet quenching as determined with a Mineralite lamp. Shaded areas represent radioactivity as determined by radioautography.

FIG. 7. Hydrolysis of Pa*-labeled PAPS by 3’nucleotidase. PAPS was prepared and chromatographed on Dowex 1 as described in the text, except that ATP labeled in the two terminal phosphate groups with Pss (400 rmoles, 0.5 mc.) was used (cf. “Materials and methods”). A sample of the radioactive PAPS fraction was digested with 3’-nucleotidase (18). Aliquots of the original solution and of the digested sample were used for electrophoresis in 0.03 M citrate, pH 5.9. Outlined areas represent ultraviolet quenching. Stippled areas represent radioactivity as determined by radioautography.


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846 ACTIVE SULFATE

natural adenosine V-phosphosulfate, but not PAPS, are attacked by bull semen 5’-nucleotidase, giving adenosine, inorganic phosphate, and inorganic sulfate. They also both serve equally well as substrates for the enzymes ATP-sulfurylase and APS-kinase that catalyze the two-step synthesis of PAPS (20).

As is shown in Fig. 7, labeled PAPS is formed when the activating system is incubated with ATP labeled with P32 in the two terminal phosphate groups. Treatment of this P3*-labeled active sulfate fraction with 3’-

0 ATP

0 ADP 4?D

AMP

OR/G/N - STANDARDS BEFORE AFTER3HRS.

INCUBATION

FIG. 8. Periodate reaction of PAPS fraction after treatment with 3’-nucleotidase. The electrophoresis pattern in Fig. 7 was sprayed with periodate, treated with SOz, and then sprayed with Schiff’s reagent according to the method of Buchanan, Dekker, and Long (23). Outlined areas represent ultraviolet quenching. Shaded areas represent the purple color, indicating a positive periodate reaction.

nucleotidase releases the label as inorganic phosphate and leaves unlabeled APS. These data suggested early that the phosphate in the 3’-position is derived by a kinase type transfer from the terminal phosphate of ATP.

As expected, neither PAP nor PAPS reacts with periodate. As shown in Fig. 8, treatment with 3’-nucleotidase leads to the formation of 5’-AMP and adenogine 5’-phosphosulfate, both of which are periodate-positive compounds. The fact that the adenosine phosphosulfate gives a positive periodate reaction indicates that the 2’,3’-hydroxyl groups are free and that the sulfate group must be bound either to the 5’-phosphate or another group of the 5’-adenylic acid. In this connection the reversal of adenosine


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phosphosulfate with pyrophosphate to form ATP and sulfate (20) gives unequivocal evidence that the sulfate is bound to the 5’-phosphate group.

The ultraviolet absorption spectra of the active sulfate fraction and of PAP (6) are identical with those of adenosine and crystalline ATP. This is further evidence that the sulfate group is not bound to the ammo group of the adenine nucleus.

o 4O%PAPS-60% PAP l AFTER ACID TREATMENT

EOUMALENTS OF HCI ADDED

FIG. 9

2 4 6 8 TIME IN HOURS FIG. 10

FIG. 9. Titration of the PAPS fraction before and after acid treatment. The PAPS fraction was prepared as described in the text by chromatography on Dowex 1. Analysis showed that 40 per cent of the adenosine was present as PAPS and that the rest could be accounted for as PAP. The sample (2 ml., 11.8 rmoles of adenosine) was adjusted to pH 8 and then titrated with 0.1 N HCI. After reaching pH 5, the sample was made 0.1 N with respect to HCl and allowed to stand for 30 minutes at 37”. It was cooled to room temperature and then adjusted to pH 8 with CO*-free 2 N KOH. It was again titrated with 0.1 N HCl to pH 5. A water blank treated in the same way as the sample showed no appreciable titration between pH 5 and 8.

FIG. 10. Formation of PAP by hydrolysis of coensyme A. The reaction mixture contained the following: 2.5 ml. of M Tris, pH 7.5; 0.3 ml. of Y MgCl,; 1 ml. of Crola- Zus adumanteus venom extract prepared according to Wang et al. (18); and 5 mg. of CoA obtained from C. H. Boehringer and Son. Samples (0.5 ml.) were heated for 2 minutes in boiling water to stop the reaction. Appropriate aliquots of the heated samples were used for CoA and PAP determinations.

Titration Data-Fig. 9 shows a typical titration curve for a fraction containing 40 per cent PAPS and 60 per cent PAP, recovered from Dowex 1. Only the secondary phosphate dissociations were titrated since the range for the titration was limited to pH 5 to 8. The shaded area repre- sents the titration of the Q/-phosphate group present in both PAP and PAPS. Since the preparation was 60 per cent PAP and only 40 per cent PAPS, the 5’-phosphate group of PAP accounts for another 0.6 equivalent of acid between pH 5 and 8. Hydrolysis of the active sulfate by very mild acid treatment leads to the appearance of 0.4 additional equivalent


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848 ACTIVE SULFATE

of secondary phosphate dissociation as would be expected after hydrolysis of a phosphosulfate. Similar data have been obtained for adenosine 5’- phosphosulfate. In this case there is no background titration due to the presence of the 3’-phosphate group.

Formation of PAP by Hydrolysis of Coenzyme A-In another publication from this laboratory, Gregory and Lipmann (6) have shown that the reaction between PAPS and p-NP is reversible, and that PAP may be assayed by measuring the rate of the PAP-dependent transfer of sulfate from NPS to phenol, catalyzed by phenol sulfokinase. If the structure of PAPS as formulated above is correct, then PAP should be formed when CoA is decomposed at the pyrophosphate bridge. Fig. 10 shows the results of an experiment in which CoA was hydrolyzed with an extract of snake venom according to the method of Wang et al. (18). Aliquots of the incubation mixture were taken and the enzymatic reaction was stopped by heating for 2 minutes at 100”. Samples of the heated solution were taken for PAP (6) and CoA assays. CoA was determined by the arseno- lysis of acetyl phosphate in the presence of transacetylase (21). As can be seen, the CoA preparation used (from C. H. Boehringer and Son) has a high concentration of PAP to begin with. Fig. 10 shows that the snake venom hydrolyzes CoA with the expected formation of PAP as determined with the phenol sulfokinase assay and also shows the equivalence of CoA disappearance and PAP appearance.

These data confirm the 3’,5’diphosphoadenosine structure of PAP. Gregory and Lipmann (6) have shown that 2’ ,5’diphosphoadenosine prepared by the hydrolysis of TPN is inactive in the PAP assay. The preparation and assay of CoAderived 3’) 5’diphosphoadenosine by an independent method have recently been described by Wang (22).

DISCUSSION

The isolation of the active sulfate confronted us with the fact that in addition to a phosphate in 5’ position, which carries the activated sulfate in an anhydride link, the compound contained another phosphate linked separately to adenosine. The presence of such a second separately linked phosphate was unexpected and the significance of an additional phosphorylation is only gradually being understood as the studies on the enzyme sequence in the biosynthesis of active sulfate have progressed (20). The more detailed report on this is now in preparation. It may be stated briefly here that a second phosphorylation seems to serve the purpose of a fixation of a thermodynamically unfavorable situation by input of additional energy t(hrough the essentially irreversible fixation of the second phosphate, derived from a second mole of ATP.

Our main comment shall be concerned with the chemical evidence for the position of the various groups in the molecule.


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With ATP being an adenosine 5’-triphosphate, the elimination of pyrophosphate, which was recognized early (1) as part of the sulfate activation reaction, indicated that the sulfate should be bound through an anhydride link to the proximal phosphate in the 5’ position. Before discussing the further confirmation of this formulation, we will first sum- marize the evidence for the position of the second phosphate, yielding directly or by exclusion confirmation of the POS link in 5’ position.

The fact that one phosphate was completely hydrolyzed by N HCl in 30 minutes at 199” indicated this to be situated in 2’ or 3’ on the ribose. The second phosphate, being acid-stable, was thus tentatively charac- terized as being in the 5’ position.

The presence of the substituent in the 2’ or 3’ position was further confirmed by the absence of periodate reaction. The final decision for the 3’ position was obtained through the use of Kaplan’s specific 3’-nucleotidase.

The fact that the dephosphorylation of the molecule with 3’nucleotidas.e yielded a sulfate-containing derivative indicated the sulfate to be connected with the 5’-phosphate. This was further confirmed by the appearance of periodate reaction after removal of the 3’-phosphate by nucleotidase, the reaction being readily given by 5’-substituted adenylic acid deriva- tives (23).

A further proof for the constitution was obtained by the use of ATP marked with Pa in the two terminal phosphates, APp1P32Ppp, in the enzymatic formation of PAPS. This yielded PaAP%, as indicated by libera- tion of Pn inorganic phosphate with 3’-nucleotidase, leaving cold APS. This observation made it rather probable that the 3’-phosphate originated by an independent enzymatic step from a second mole of ATP.

The unusual binding of sulfate by an anhydride link to phosphate needs still some further comment.

(1) An appearance of periodate reaction after removal of the 3’-phosphate excluded the possibility of the sulfate being linked directly to a hydroxyl in the ribose.

(2) The possibility that sulfate might be attached to the amino group of the sdenosine is fairly well excluded by the ultraviolet spectrum which is identical with that of adenosine 5’-phosphate. In all cases studied, a substitution of the amino group causes a shit in the ultraviolet toward the visible (24, 25).

(3) Positive confirmation of the linking was obtained through electroti- tration by showing that 1 equivalent of secondary phosphate is liberated by mild hydrolysis of the sulfate.

Independent confirmation of our structure was furnished through the synthesis of adenosine 5’-phosphosulfate by Baddiley et al. (19) from adenosine 5’-phosphate and the pyridine complex of SO1. This procedure


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850 ACTIVE SULFATE

might be considered as speci6c for an attack on the 5’-phosphate (cf. Avison (26)). The identity of the synthetic compound with the product of 3’dephosphorylation with nucleotidase has been mentioned already. Furthermore, recently Baddiley and his collaborators have succeeded in synthesizing the adenosine 3’-phosphate-5’-phosphosulfate from synthetic or natural PAP, by using a procedure analogous to the one used for the synthesis of adenosine 5’-phosphosulfate (27). The proposed structure is further confirmed by identification of the sulfate-free residue of active sulfate with the product obtained from CoA by hydrolysis at its pyrophosphate bridge. This hydrolysis product had been earlier identified by Wang et al. (18) as adenosine 3’,5’-diphosphate.

Adenosine 3’ ,5’diphosphate has previously been identified as an end group in ribonucleic acid (28).

SUMMARY

Procedures are described for the separation in liver extracts of the sulfate-activating enzyme system from phenol sulfokinase, the enzyme responsible for the transfer of sulfate from the active sulfate to nitrophenol.

The purified phenol sulfokinase was used for the assay of active sulfate in various states of purity, in particular, in the presence of adenosine triphosphate plus sulfate. The sulfate-activating system was used for the preparation of active sulfate in quantities of 50 rmoles.

Active sulfate was then isolated by Dowex 1 chromatography, with use of mixtures of formic acid and ammonium formate for the separation from other nucleotides.

The analysis of the compound showed it to contain 2 moles of phosphate for 1 mole of adenosine and sulfate. The compound was identified as adenosine 3’-phosphate-5’-phosphosulfate.

BIBLIOGRAPHY

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Phillips W. Robbins and Fritz LipmannACTIVE SULFATE

ISOLATION AND IDENTIFICATION OF

1957, 229:837-851.J. Biol. Chem.

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by phillips w. rcbbinst and generallowing: 1000 mmoles of imidasole*hcl, ph 7; 100 pmoles of...

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